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DOI: 10.3389/fnins.2018.00080

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Citation for published version (APA): Bridi, J. C., & Hirth, F. (2018). Mechanisms of - Induced Synaptopathy in Parkinson's Disease. Frontiers in Neuroscience, 12, 80. DOI: 10.3389/fnins.2018.00080

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Download date: 16. Jun. 2018 REVIEW published: 19 February 2018 doi: 10.3389/fnins.2018.00080

Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson’s Disease

Jessika C. Bridi and Frank Hirth*

King’s College London, Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, London, United Kingdom

Parkinson’s disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be

Edited by: the most toxic species, with their accumulation at presynaptic terminals affecting Mark P. Burns, several steps of release. First, high levels of α-Syn alter the size Georgetown University, United States of pools and impair their trafficking. Second, α-Syn overexpression Reviewed by: Fu-Ming Zhou, can either misregulate or redistribute of the presynaptic SNARE complex. University of Tennessee Health This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at Science Center, United States the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, Jiajie Diao, University of Cincinnati, United States accompanied by a decrease in AZ levels. Furthermore, α-Syn overexpression *Correspondence: reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. Frank Hirth These presynaptic alterations mediated by accumulation of α-Syn, together impair [email protected] neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed

Specialty section: throughout the brain and enriched at presynaptic terminals, DA neurons are the most This article was submitted to vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, Neurodegeneration, a section of the journal evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes Frontiers in Neuroscience responsible for its synthesis. In addition, α-Syn is able to interact with and reduce Received: 05 December 2017 the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly Accepted: 01 February 2018 contributes to the formation of toxic α-Syn oligomers. Together these data suggest a Published: 19 February 2018 vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic Citation: Bridi JC and Hirth F (2018) dysfunction and impaired neuronal communication, ultimately causing synaptopathy and Mechanisms of α-Synuclein Induced progressive neurodegeneration in Parkinson’s disease. Synaptopathy in Parkinson’s Disease. Front. Neurosci. 12:80. Keywords: Parkinson’s disease, synapse, SNARE complex, active zone, dopamine, α-synuclein, synaptopathy, doi: 10.3389/fnins.2018.00080 neurodegeneration

Frontiers in Neuroscience | www.frontiersin.org 1 February 2018 | Volume 12 | Article 80 Bridi and Hirth Mechanisms of α-Synuclein Induced Synaptopathy

INTRODUCTION including constipation, olfactory dysfunction (hyposmia), sleep disturbance, obesity and depression (Figure 1C; Hawkes et al., Parkinson’s disease (PD) is the second most common 2010; Kalia and Lang, 2015; Mahlknecht et al., 2015). During neurodegenerative disorder after Alzheimer’s disease (AD) the prodromal phase of PD and PD-related disorders, which (Kalia and Lang, 2015). The pathological hallmarks of PD precedes degenerative cell loss, the expression levels of a range are intracellular proteinaceous inclusions called Lewy bodies of proteins involved in synaptic transmission are altered in (LB) and Lewy neurites (LN) that are predominantly formed the prefrontal and cingulate cortex, and SN (Dijkstra et al., of misfolded and aggregated forms of the presynaptic protein 2015; Bereczki et al., 2016; Table 1), suggesting that both non- α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons motor and motor symptoms are caused by impaired synaptic in the substantia nigra (SN) (Spillantini et al., 1997; Lang communication. This data indicates that neurodegeneration in and Lozano, 1998a,b). Loss of DA neurons in the SN leads to PD is a dying back-like phenomenon which starts at synaptic marked decrease of dopamine levels in synaptic terminals of the terminals in the striatum and progresses along the nigrostriatal dorsal striatum (Figures 1A,B), ultimately leading to a loss of pathway, ultimately affecting homeostasis and survival of DA cell the nigrostriatal pathway (Cheng et al., 2010). The reduction bodies in the SN (Hornykiewicz, 1998; Calo et al., 2016; Caminiti of striatal dopamine triggers a range of motor symptoms et al., 2017). It is due to these early-onset synaptic alterations including bradykinesia, uncontrollable tremor at rest, postural observed prior to DA loss, PD has also been classified as impairment, and rigidity which together characterize PD as a a synaptopathy (Brose et al., 2010; Schirinzi et al., 2016). movement disorder. The majority of PD cases are sporadic with unknown cause. The onset of PD, however, is considered to commence However, familial cases with autosomal dominant or recessive at least 20 years prior to detectable motor abnormalities, Mendelian inheritance have revealed fundamental insights into when a variety of non-motor symptoms can be observed pathogenic mechanisms underlying PD (Keane et al., 2011; (Hawkes et al., 2010; Kalia and Lang, 2015; Mahlknecht et al., Massano and Bhatia, 2012; Kalia and Lang, 2015). The most 2015). This period is referred to as the prodromal phase commonly identified genetic linked to heritable PD where patients experience a range of non-motor symptoms were found in the SNCA and LRRK2, responsible for

FIGURE 1 | Midbrain dopaminergic neurons are specifically vulnerable in Parkinson’s disease. (A) The predominant symptoms of Parkinson’s disease (PD) are caused by loss of dopaminergic (DA) neurons in the substantia nigra (SN). According to the dying-back hypothesis, the degeneration of DA neurons is preceded by dysfunction and in turn degeneration of the nigrostriatal pathway, which innervates the caudate nucleus and the putamen that together form the striatum. (B) Compared to healthy controls (left), nigrostriatal degeneration results in the depletion and ultimate loss of the neurotransmitter dopamine on synaptic terminals of striatal neurons (right). (C) The resulting motor symptoms, among others, are usually diagnosed when approximately 30–60% of striatal DA neurons are already lost. However, PD patients can experience non-motors symptoms 20 years before the onset of motor abnormalities in the so-called prodromal phase; these include olfactory dysfunction, sleep disturbances and depression.

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TABLE 1 | Alterations of presynaptic proteins seen with α-Syn related proteinopathy in PD and DLB patients and rodent animal models.

Protein Modification Species Assay system References

Synapsin 1 Down Human Post-mortem tissue from DLB patients Scott et al., 2010 Rat BAC rat model overexpressing the full-length human SNCA Kohl et al., 2016 Mouse Hippocampal neurons overexpressing human WT α-Syn:GFP Scott et al., 2010 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 2 Down Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Mouse Primary neuronal culture treated with α-Syn pre-formed fibrils Volpicelli-Daley et al., 2011 Synapsin 3 Down Human iPSCs from patients harboring A53T Kouroupi et al., 2017 Up Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 Down Human Post-mortem tissue from DLB patients Kramer and Schulz-Schaeffer, 2007 Mouse Overexpression of human α-Syn under the mThy1 promoter Games et al., 2014 Up Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 CSPα Down Mouse Primary neuronal culture treated with α-Syn pre-formed fibrils Volpicelli-Daley et al., 2011 SV glycoprotein 2 Down Human iPSCs from patients harboring A53T mutation Kouroupi et al., 2017 Rab3A/Rab3 Down Human iPSCs from patients harboring A53T mutation Kouroupi et al., 2017 Human Post-mortem tissue from PDD and DLB patients Bereczki et al., 2016 Rat Nigral injection of AAV2-A53T-α-Syn Chung et al., 2009 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 VAMP-2 Down Mouse Hippocampal neurons overexpressing human WT α-Syn:GFP Scott et al., 2010 Mouse Primary neuronal culture treated with α-Syn pre-formed fibrils Volpicelli-Daley et al., 2011 Misloc. Human Post-mortem tissue from PD patients (striatum-putamen and Garcia-Reitböck et al., 2010 external globus pallidus) Up Mouse Truncated human α-Syn Garcia-Reitböck et al., 2010 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 SNAP-25 Down Human Post-mortem tissue from PDD and DLB patients Bereczki et al., 2016 Mouse Primary neuronal culture treated with α-Syn pre-formed fibrils Volpicelli-Daley et al., 2011 Misloc. Human Post-mortem tissue from PD patients (striatum-putamen and Garcia-Reitböck et al., 2010 external globus pallidus) Mouse Truncated human α-Syn Garcia-Reitböck et al., 2010 Up Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Syntaxin-1/Syntaxin Down Human Post-mortem tissue from DLB patients Kramer and Schulz-Schaeffer, 2007 Rat Nigral injection of AAV2-A53T-α-Syn Chung et al., 2009 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Misloc. Human Post-mortem tissue from PD patients (striatum-putamen and Garcia-Reitböck et al., 2010 external globus pallidus) Mouse Truncated human α-Syn Garcia-Reitböck et al., 2010 Synaptotagmin 1 Up Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Synaptotagmin 2 Up Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 Complexin 1 Down Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Complexin 2 Down Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Synphilin 1 Down Human Post-mortem tissue from PD patients (Braak stages 1–2) Dijkstra et al., 2015 Munc18-1 Up Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Munc13-1 Down Mouse Overexpression of human WT α-Syn Nemani et al., 2010 Piccolo Down Mouse Hippocampal neurons overexpressing human WT α-Syn:GFP Scott et al., 2010 RIM3 Down Rat BAC rat model overexpressing the full-length human SNCA Kohl et al., 2016 Amphiphysin Down Mouse Hippocampal neurons overexpressing human WT α-Syn:GFP Scott et al., 2010

Frontiers in Neuroscience | www.frontiersin.org 3 February 2018 | Volume 12 | Article 80 Bridi and Hirth Mechanisms of α-Synuclein Induced Synaptopathy an autosomal-dominant forms of PD, and in Parkin, PINK1, together with β-Syn and γ-Syn belong to the synuclein protein DJ-1, and ATP13A2 which account for PD with autosomal family (Goedert, 2001; Marques and Outeiro, 2012). The three recessive mode of inheritance (Klein and Westenberger, 2012; Synuclein proteins are 55–62% identical in sequence and have Ferreira and Massano, 2017). PD-related mutations in PINK1 similar domain organization (Goedert, 2001); all three are highly and Parkin and the functional interaction of the two proteins led expressed in the human brain (Goedert, 2001). Additionally, to the identification of mitochondrial dysfunction as one of the α-Syn and β-Syn share the same subcellular distribution at major pathogenic pathways underlying PD (reviewed in Exner presynaptic terminals in neurons (Jakes et al., 1994; Goedert, et al., 2012; Pickrell and Youle, 2015). Additionally, PD-related 2001). However, α-Syn is the only protein of the synuclein family mutations in Glucocerebrosidase (GBA), SCARB2 and ATP13A2 to be found in LB and to be implicated in PD pathogenesis established lysosomal storage dysfunction as a second pathogenic (Goedert et al., 2017). pathway that also contributes to PD etiology (reviewed in Hardy, Several dominant-inherited single point mutations in SNCA 2010; Gan-Or et al., 2015). have been identified in families that develop early onset However, among the identified PD-related genes, SNCA PD (Polymeropoulos, 1997; Krüger et al., 1998; Zarranz et al., encoding the presynaptic protein α-Syn remains the most 2004; Appel-Cresswell et al., 2013; Lesage et al., 2013; Pasanen potent culprit underlying PD. Accumulated α-Syn is the main et al., 2014). Subsequent studies identified multiplications of the component of LB, and together with genome-wide association SNCA gene (Singleton, 2003; Farrer et al., 2004) and SNCA studies it has been shown to have central pathogenic role in polymorphisms which are associated with high risk of developing both familial and sporadic PD (Satake et al., 2009; Simón- the disease (Satake et al., 2009; Simón-Sánchez et al., 2009). In Sánchez et al., 2009). Yet despite recent progress, the pathogenic line with dosage-related α-Syn pathology, higher doses of α- mechanisms underlying α-Syn related PD are only starting to Syn observed in patients with duplication and triplication of the emerge. Here we provide a focussed review emphasizing current SNCA gene locus directly correlate with cognitive decline, motor knowledge on synaptic function and the various mechanisms of and non-motor symptoms and severity of neurodegenerative α-Syn induced synaptopathy, and its role in the early stages of PD phenotypes (Venda et al., 2010). This data is strong evidence progression. that dysfunction and accumulation of α-Syn plays a key role in both familial and sporadic forms of PD. In addition to LB inclusions, α-Syn has been shown to accumulate in (Braak ACCUMULATION OF α-SYNUCLEIN: THE et al., 1999) and in the presynaptic terminal (Kramer and Schulz- PATHOLOGICAL HALLMARK OF PD Schaeffer, 2007), suggesting these pathogenic accumulations are a key trigger of onset and formation of PD-related synaptopathy α-Syn is a small soluble cytoplasmic protein of 140 amino (Stefanis, 2012; Schirinzi et al., 2016) (Box 1). acid; its main protein domains comprise an amphipathic region, In the healthy brain and central nervous system (CNS), wild non-amyloid-β component (NAC) domain and an acidic tail type α-Syn is available in its native conformation as soluble (Figure 2A; Maries et al., 2003; Venda et al., 2010). α-Syn monomers. These are thought to mediate its physiological

FIGURE 2 | The presynaptic protein α-Synuclein is a pathological seed for Parkinson’s disease formation. (A) α-Synuclein (α-Syn) is a small soluble cytoplasmic protein of 140 amino acid encoded by the SNCA gene; its main protein domains comprise an N-terminal amphipathic region, a non-amyloid-β component (NAC) domain and a C-terminal acidic tail. Several dominant inherited missense mutations have been identified in the amphipathic region causing early-onset PD, whereas the NAC domain has been implicated in α-Syn aggregation. (B) Under normal physiological conditions, α-Syn monomers function in a dynamic equilibrium between a soluble and a membrane-bound state. Under cellular stress and in disease-relation conditions, α-Syn monomers can interact leading to oligomers that buffer the formation of protofibrils, ultimately causing the formation of amyloid-β sheet fibrils that aggregate into Lewy bodies (LB).

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BOX 1 | α-Syn-mediated synaptopathy - open questions and future directions.

Progress over the past two decades revealed compelling evidence that one of the major sites of α-Syn-mediated toxicity are presynaptic terminals. Once initiated, the resulting neuronal dysfunction can lead to both cell intrinsic and cell-extrinsic toxicity. Cell-intrinsic mechanisms cause axonal degeneration which progresses toward the cell body, eventually leading to dying-back like neurodegeneration. Cell-extrinsic mechanisms cause transmission of α-Syn oligomers along synaptic connections and the ultimate progression of synucleopathy throughout the patient brain. Neither of these mechanisms are fully understood, nor how α-Syn oligomerizes in the first place, thus precluding the development of efficient therapeutic treatments. To progress toward therapy, several open questions need to be addressed.

Conformational changes of α-Syn α-Syn accumulates in synapses and axons and forms neuronal inclusions. What triggers α-Syn self-assembly and in turn the formation of toxic species of misfolded α-Syn? How do different types of oligomers serve as template for fibril formation of α-Syn? There is evidence that the formation of fibrils, rather than toxic species themselves, mediates α-Syn toxicity (Lashuel et al., 2013). Which aspect of this process is so pathogenic to neurons? It also remains to be shown whether α-Syn may form various types of oligomeric states with different toxicity. To address these questions in a systematic way, first and foremost reliable markers are required to monitor, both in vitro and in vivo, the different and progressive conformational changes of α-Syn and their impact on toxicity. α-Syn acts as a chaperone for the presynaptic SNARE complex. It controls assembly, maintenance and distribution of this highly reactive complex of proteins. Can this environment be responsible for triggering the pathologic cascade to form α-Syn oligomers and fibrils? The increased stability and longer half-life of mutant forms of α-Syn, such as A30P and A53T, increase their potential of aggregation (Bennett et al., 1999; Cuervo et al., 2004). In addition, failure of proteasomal degradation and autophagy in PD have been linked to high levels of α-Syn. Together, these factors can favor the accumulation of pathologic α-Syn. Small molecule and compound screens together with in vivo genetic screens in animal models are necessary to identify mechanisms and molecules that can abrogate, hamper or at least delay misfolding of α-Syn, and thus progression of disease.

Accumulation of α-Syn and synaptic defects What is the threshold concentration of toxic α-Syn species, such as oligomers and fibrils, to trigger biochemical and cellular alteration that lead to neuronal dysfunction? Oligomeric forms of α-Syn are considered the most toxic species at the synapses (Ingelsson, 2016) and their structure appears different between mutant forms and wild type α-Syn (Tosatto et al., 2015). This suggests the severity of pathology may not only relate to the abundance and formation of oligomers but also to their structural and functional properties. It remains to be shown whether distinct oligomeric α-Syn species, and their way of formation, exert differential toxic mechanisms that specifically impair the presynaptic terminal and neurotransmission. Do oligomeric and fibrils of α-Syn cause a dominant negative effect in synaptic function? Can α-Syn oligomers, for instance, reduce the activity of its synaptic partners and neuronal signaling pathways? The effect of different lengths and concentrations of α-Syn oligomers on SNARE-mediated membrane fusion and docking are one way to measure toxicity, as previously shown in vitro (Choi et al., 2013; Lai et al., 2014). In vitro protein-reconstituted systems are necessary to test the effects of different toxic α-Syn species on fast Ca2+-triggered neurotransmitter release and their in vivo toxicity in synaptic transmission.

α-Syn transmission and transcellular progression of disease Several studies using animal models have given strength to Braak’s hypothesis for α-Syn propagation within interconnected brain regions (reviewed in Oueslati et al., 2014). However, the mechanisms underlying α-Syn transmission remain poorly understood. Which α-Syn toxic species are “seeding” for trans-synaptic transmission? What is the minimum concentration of toxic α-Syn required to trigger transmission? Can extracellular α-Syn and exosomes be efficiently cleared by pharmacological or gene therapy approaches without affecting the intracellular endogenous α-Syn? To address these questions, in vivo systems to allow monitoring of α-Syn transmission are required. For example, Drosophila melanogaster has been successfully used to study transcellular spreading of human huntingtin (Htt) (Babcock and Ganetzky, 2015). This model system has opened avenues for unbiased whole genome screening of cellular mechanisms and pharmacological interventions of Htt transmission. α-Syn transmissibility could also be studied in brain organoids derived from induced pluripotent stem cells (iPSCs) of healthy individuals and patients harboring α-Syn mutations. Although an in vitro culture, organoids resemble the in vivo architecture, functionality, and genetic signature of original tissues (Dutta et al., 2017). Moreover, organoids develop neuronal networks that can be modulated by sensory stimulation (Quadrato et al., 2017). Hence organoids can be used to study α-Syn propagation along interconnected neuronal networks.

function in presynaptic terminals (Burré, 2015). Although LB are The carriers of A30P and A53T α-Syn point mutations are also characterized by β-sheet aggregated fibrils of α-Syn (Spillantini more prone to α-Syn aggregation and toxicity, hence correlating et al., 1997; Rodriguez et al., 2015), in vitro studies have shown with increased vulnerability and earlier onset of PD formation in that the formation of oligomers, not fibrills, are a distinct SCNA mutant carriers (Li et al., 2001; Ingelsson, 2016). hallmark of α-Syn mutations such as A30P and A53T, which Aggregated and oligomeric α-Syn are also implicated in prion- are known to cause early-onset familial PD (Conway et al., like transmission of progressive PD pathology along distant but 2000). The NAC domain of α-Syn is a hydrophobic sequence connected brain regions (Spillantini et al., 1997; Jucker and formed of 12 amino acids which forms the core part of the Walker, 2013). The accumulation of α-Syn in LB undergo an α-Syn protein and is key for its transformation from soluble ascending pattern of distribution, from the lower brainstem monomer to oligomers, into protofibrils and finally aggregating and olfactory bulb into the limbic system and ultimately to fibrils (Figure 2B; Giasson et al., 2001). In vitro studies performed the neocortex (Braak et al., 2003). Several hypotheses attempt by Narhi and colleagues demonstrated that wild type, as well to address the cellular mechanisms underlying progressive α- as mutant A30P and A53T α-Syn can form insoluble β- Syn transmission spreading throughout these brain regions sheet structured fibrillar aggregates at physiological temperature (Longhena et al., 2017). Few studies have supported that cell- (Narhi et al., 1999). Importantly, mutant forms of α-Syn promote to-cell transmissibility of α-Syn occurs at the synapses, through fibril formation more rapidly than the wild type α-Syn protein. physiological processes such as exocytosis and endocytosis or due

Frontiers in Neuroscience | www.frontiersin.org 5 February 2018 | Volume 12 | Article 80 Bridi and Hirth Mechanisms of α-Synuclein Induced Synaptopathy to synaptic degeneration (Frost and Diamond, 2009; Guo and considered to be the first step followed by active deconstruction Lee, 2014). Although the underlying mechanisms so far remained of axons and loss of neuronal connectivity, eventually leading elusive, there is strong evidence that progressive transmission to the death of neuronal perikarya (Figure 3; Scott et al., 2010; correlates with severity of the disease (Hawkes et al., 2010) Lu et al., 2014; Morales et al., 2015; Schulz-Schaeffer, 2015; Calo (Box 1). et al., 2016; Grosch et al., 2016; Tagliaferro and Burke, 2016; Bae and Kim, 2017; Fang et al., 2017; Kouroupi et al., 2017; Roy, WHERE DOES PD PATHOLOGY START? 2017). This succession of events suggests that neural death in PD is initiated at synaptic terminals and progresses proximally Several neurodegenerative diseases exhibit early impairment toward neural cell bodies in a dying back-like manner. of synaptic function (Bae and Kim, 2017). This often occurs In line with this hypothesis, almost 20 years ago Hornykiewicz concomitantly with the manifestation of cognitive symptoms, suggested that formation of PD starts by affecting axons in with a neuronal degeneration emerging at later stages of the dorsal striatum prior to degeneration of DA neurons in disease (Milnerwood and Raymond, 2010; Schulz-Schaeffer, the SN (Hornykiewicz, 1998). However, this hypothesis only 2010; Picconi et al., 2012). Thus, synaptic dysfunction is recently gained strong support by a range of pathological and

FIGURE 3 | α-Syn accumulation in presynaptic terminals causes synaptopathy ultimately leading to dying back-like neurodegeneration. (A) Under physiological conditions, α-Syn functions as monomers at the presynaptic terminals in synaptic transmission. (B) Formation of toxic α-Syn species, such as oligomers and fibrils, have been shown to play a pivotal role in PD pathogenesis. These toxic species accumulate at the presynaptic terminal, leading to altered levels of proteins involved in synaptic transmission, ultimately causing synaptic dysfunction. (C) As a result of toxic α-Syn accumulation, affected synapses will undergo a process of active deconstruction leading to loss of neuronal connections and subsequent death of the neuronal perikarya.

Frontiers in Neuroscience | www.frontiersin.org 6 February 2018 | Volume 12 | Article 80 Bridi and Hirth Mechanisms of α-Synuclein Induced Synaptopathy molecular data. The first evidence that axons were affected in The presynaptic nerve terminal is a specialized secretory PD came from the pioneer study performed by Braak et al. machinery that releases by synaptic vesicle (1999). They were able to demonstrate that extensive and very (SV) exocytosis in response to an action potential. It is at the thin α-Syn inclusions were not only present in LB inclusions presynaptic terminal where α-Syn is hypothesized to play its at the neuron soma but also present in axonal processes physiological or pathological role (Katz, 1969; Chandra et al., (Braak et al., 1999). More recent studies found α-Syn localized 2005; Burré, 2015). By receiving an action potential, calcium within axonal dystrophic neurites in the striatum of patients (Ca2+) channels open, thereby allowing influx of Ca2+ into with Alzheimer’s disease (AD) and PD (Duda et al., 2002), the presynaptic terminal (Südhof, 2013b). This Ca2+ influx as well as in multiple system atrophy (MSA) and dementia promotes the assembly of a molecular machinery composed with Lewy body (DLB) (Galvin et al., 1999). The localization mainly of the Ca2+ sensor synaptotagmin and the soluble of α-Syn was further studied through the development of a NSF-attachment protein receptor (SNARE) complex, including technique called paraffin-embedded tissue (Kramer and Schulz- VAMP-2, SNAP-25, and Syntaxin-1 (Südhof, 2013a). Ultimately, Schaeffer, 2007). This method allowed for the detection of this molecular machinery will trigger SV trafficking from the abundant α-Syn micro-aggregates in the neuropil rather than closest pool, the readily releasable pool (RRP), to be tethered, in the cell body of DLB patient brains. Furthermore, Kramer docked and fused to the presynaptic plasma membrane at a and Schulz-Schaeffer showed the abundance of synaptic α- defined region of the presynaptic terminal called the active Syn micro-aggregates exceeded the amount of α-Syn aggregates zone (AZ) (Figure 4), for subsequent release of neurotransmitter in LB or LN by one to two orders of magnitude. Their in the synaptic cleft (Südhof, 2000, 2012, 2013b; Alabi and filtration technique revealed 50–92% of α-Syn micro-aggregates Tsien, 2012). In addition to the SNARE complex, a subfamily were found entrapped within the presynaptic terminal. This of highly conserved small GTPases called Rab are implicated in result correlated with the striking downregulation of presynaptic intracellular trafficking of vesicles and the recruitment of SV in proteins like syntaxin and synaptophysin, and postsynaptic the AZ (Binotti et al., 2016). proteins such as PSD95 and drebrin (Kramer and Schulz- The AZ matrix is composed of several proteins including Schaeffer, 2007). RIM, ELKS (also known as ERC/CAST/Rab6IP2), Munc13, This new mechanistic insight exploring synaptic deficits as a Bassoon/Piccolo, Rim-BP, and Liprin-α (Schoch and starting point in α-Syn related pathology is in agreement with Gundelfinger, 2006; Südhof, 2012; Wang et al., 2016). The previous findings, where α-Syn aggregates were observed in AZ is a highly organized structure restricted to the plasma terminals preceding the formation of LB in DLB and correlating membrane region, opposite to the postsynaptic density marked with cognitive impairment (Marui et al., 2002). Moreover, similar by postsynaptic density protein 95 (PSD95). Functionally, AZ to DLB, PD patient data demonstrates that these individuals proteins dock SVs to be fused with the plasma membrane and experience first locomotor symptoms once 50–60% of DA release their neurotransmitter content into the synaptic cleft striatal terminals have already been lost, while the loss of DA (Figure 4; Wang et al., 2016). After exocytosis, the SV membrane neurons in the SN is believed to be only around 30% (reviewed is retrieved from the synaptic cleft by endocytosis, either via in Burke and O’Malley, 2013). These observations have been clathrin-dependent slow or clathrin-independent fast modes of independently confirmed by positron emission tomography; PD endocytosis (Saheki and De Camilli, 2012). The SV are locally patients in early stages of the disease show extensive axonal loaded with neurotransmitter, hence being recycled to allow damage and loss of nigrostriatal pathway connectivity (Caminiti another round of exo-endocytotic membrane cycling (Südhof, et al., 2017). This data suggests that α-Syn pathology is abundant 2004; Gross and von Gersdorff, 2016). This mechanism allows in presynaptic terminals and axons, in line with the observation the neurons to sustain a high firing rate without depletion of the that its normal localization is predominantly in presynaptic SV pools (Saheki and De Camilli, 2012; Gross and von Gersdorff, terminals (Figure 3). Yet little is known how early accumulation 2016). of toxic α-Syn species impairs synaptic homeostasis and function, Deficiencies in synaptic transmission have been observed ultimately leading to DA neurodegeneration in PD. in response to both knock down or overexpression of α-Syn, suggesting that it has a role in the regulation of neurotransmitter release, synaptic function and homeostasis (Box 1) (Kahle et al., THE JANUS FACE OF PRESYNAPTIC 2000; Lee et al., 2008; Zhang et al., 2008; Lashuel et al., α-SYN: PHYSIOLOGY AND PATHOLOGY 2013; Vargas et al., 2017). Under physiological conditions, α- Syn is found at the presynaptic terminal (Maroteaux et al., Synapses are the intercellular junctions between a presynaptic 1988) and exists in a dynamic equilibrium between a soluble neuron and a postsynaptic cell (Südhof, 2012). They are used and a membrane-bound state (Burré, 2015). Specifically, α-Syn to transmit signal between neurons in the CNS via exocytosis is localized to presynaptic boutons due to its preference for of neurotransmitter allowing an organized flux of information membranes with high curvature (Middleton and Rhoades, 2010; through the brain (Lepeta et al., 2016). Synapses are composed of Jensen et al., 2011). Despite its known presynaptic localization, a presynaptic terminal, synaptic cleft and postsynaptic terminal the physiological functions of α-Syn remain poorly understood. which require a complex and tight regulation for proper In the following sections, we will discuss the evidence for neurotransmission to occur (Figure 4; Südhof and Rizo, 2011; physiological and pathological roles of α-Syn in the presynaptic Südhof, 2012). terminal.

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FIGURE 4 | The presynaptic exo-endocytotic cycle regulating neurotransmitter release is specifically affected in α-Syn-related synaptopathies. Upon an incoming action potential, calcium (Ca2+) channels become permeable to Ca2+ entry in the presynaptic terminal. This activates a molecular machinery including the SNARE complex proteins that recruit synaptic vesicles (SV) from the proximal resting and recycling (RP) pools via trafficking and tethering to form the readily releasable pool (RRP). After docking and priming, RRP vesicles undergo SNARE-mediated membrane fusion at the active zone (AZ, shaded area), ultimately leading to neurotransmitter (NT) release into the synaptic cleft. After exocytosis, the SV membrane is retrieved to the presynaptic terminal via endocytosis, to be filled with NT (NT uptake) and re-enter the exo-endocytotic cycle, thereby granting the neuron its ability to sustain high firing rates. PSD95, postsynaptic density protein-95.

α-Syn Affects Synaptic Vesicle Pools and without affecting docked vesicles at the RRP (Cabin et al., 2002). Their Trafficking in the Presynaptic In addition, the refilling of the docked vesicles by the resting Terminal pool after depletion was slower in the synapses of mice lacking The SV pools can be divided accordingly to the number of α-Syn (Cabin et al., 2002). Together this data strongly supports SVs and distance from the AZ (Südhof, 2000; Alabi and Tsien, a role of α-Syn in SV trafficking at the presynaptic terminal by 2012). The RRP is the closest in proximity to the AZ, containing modulating vesicular dynamics and maintaining the homeostasis the smallest number of vesicles and holding the highest fusion of vesicle RPs. probability. The recycling pool (RP) is located above the RRP Overexpression of wild type and mutant α-Syn in Chromaffin and has three times more vesicles than the RRP. The RP and PC12 cells revealed inhibited evoked neurotransmitter repopulates vacancies at the RRP which are often rate-limiting release (Larsen et al., 2006). This inhibition was attributed to during persistent activity and along with the RRP compose the a smaller population of RRP, suggesting that α-Syn inhibits the total recycling pool (Alabi and Tsien, 2012). Finally, the most vesicle “priming” step which is the intermediate step between distal SV pool is called the resting pool, this is the largest pool vesicle docking and fusion of synaptic vesicles at the AZ (Larsen of SVs that remain unreleased even after prolonged synaptic et al., 2006). Furthermore, overexpression of wild type α-Syn stimulation (Figure 4; Südhof, 2000; Alabi and Tsien, 2012). to similar levels observed in PD patients with duplication α-Syn has been shown to be associated with the recycling and triplication of the SNCA gene, inhibited neurotransmitter pool of SVs and its proteins. Depletion of α-Syn, through the exocytosis by affecting the size of the RP (Nemani et al., 2010; use of antisense oligonucleotides, induces a decrease in the Scott and Roy, 2012). availability of reserve synaptic vesicle pool in primary cultured Interestingly, ultrastructural analysis of the synapse in mice hippocampal neurons (Murphy et al., 2000). α-Syn knockout overexpressing human wild type α-Syn demonstrated the total (KO) mice revealed a striking deficiency of undocked vesicles number of SVs in the presynaptic terminal was not altered.

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Instead, these animals displayed reduced numbers of SVs at a synaptic vesicle docking via distinct mechanisms (Lai et al., close proximity to the AZ, whereas numbers of SVs at greater 2014). These data suggest that α-Syn may form various types of distances were increased (Nemani et al., 2010). Furthermore, oligomeric states with different toxicity, as has been shown for this study concluded that exocytotic deficits caused by α-Syn aggregated tau protein in tauopathies (Goedert, 2015). Moreover, overexpression were not only caused by reduced size of the RP, Scott and colleagues showed that cultured hippocampal neurons but also due to disturbances of SV reclustering after endocytosis from transgenic animals overexpressing human α-Syn displayed (Nemani et al., 2010). These findings demonstrate high levels of absence of endogenous synaptic proteins, including the SNARE α-Syn can trigger impairment of exocytosis via alteration of SV complex protein VAMP-2 and SV proteins amphiphysin pools in absence of neurodegeneration. and synapsin-1 (Scott et al., 2010). Neurons overexpressing fluorescent human α-Syn demonstrated marked deficits in α neurotransmitter release, in addition to reduced numbers of SVs, The SNARE Complex Related to -Syn and enlargement of the remaining SVs (Scott et al., 2010). Studies Function and Dysfunction investigating levels of synaptic proteins belonging to the SNARE The SNARE proteins are crucial to allow SVs to fuse with complex in PD, PD with dementia (PDD) and DLB post-mortem the plasma membrane at the AZ to release neurotransmitter samples also reported misregulation of proteins belonging to the (Südhof and Rizo, 2011). The subsequent exocytosis requires the SNARE complex (Kramer and Schulz-Schaeffer, 2007; Dijkstra assembly of presynaptic machinery proteins thousands of times et al., 2015; Bereczki et al., 2016, 2018). Moreover, an in vitro per minute. During each episode, the SNARE complex assembly model of α-Syn toxicity, primary neuronal cultures exposed to and disassembly gives rise to extremely reactive unfolded SNARE preformed α-Syn fibrils exhibited loss of the SNARE complex protein intermediates, exposing the presynaptic terminal to proteins VAMP2 and SNAP-25, as well as the SV proteins CSPα potentially vulnerable activity-dependent degeneration (Burré and synapsin-2 (Volpicelli-Daley et al., 2011). Volpicelli-Daley et al., 2010; Südhof and Rizo, 2011). α-Syn has been shown to and colleagues further investigated the impact of accumulation act as a chaperone to promote SNARE complex assembly and of these preformed α-Syn fibrils in neural network activity. to support its folding through direct binding to VAMP-2 and Interestingly, they found that impaired hippocampal network phospholipids on synaptic vesicles (Burré et al., 2010). activity occurred much earlier than the detected reduction in In line with these observations, α-Syn overexpression is synaptic protein levels, suggesting that α-Syn pathology can have able to rescue neurodegeneration observed in CSPα KO mice a major impact on the coordination of neuronal communication by chaperoning the assembly of the SNARE complex, while and connectivity (Box 1) (Volpicelli-Daley et al., 2011). concomitant knockout of α-Syn exacerbates the CSPα KO Conversely, a mouse model expressing human truncated α- phenotype (Chandra et al., 2005). CSPα acts as a chaperone Syn leading to α-Syn aggregates did not show changes in levels of of the SNARE complex by supporting the functional ability of synaptic proteins but rather caused age-dependent redistribution SNAP-25 to engage in a complex with Syntaxin-1 and VAMP- and accumulation of SNAP-25, Syntaxin-1, and VAMP-2 in 2 (Figure 5A; Sharma et al., 2011). The chaperone function the striatum accompanied by an age-dependent reduction in of CSPα within the SNARE complex is shared with α-Syn, dopamine release (Garcia-Reitböck et al., 2010). SNARE proteins however the mechanisms differ. α-Syn binds to VAMP-2 via its were also redistributed in the striatum and external globus C-terminal (Burré et al., 2010) which appears to be essential for α- pallidus of early-onset PD patients (Garcia-Reitböck et al., 2010). Syn mediated neuroprotection because the A30P α-Syn mutant, Taken together, evidence from transgenic mouse models and PD which is deficient in its ability to bind to membranes, failed patients demonstrate that accumulated α-Syn alters the levels to rescue CSPα KO mice (Chandra et al., 2005). This role of and/or localization of SNARE proteins at the presynaptic nerve native α-Syn in assisting SNARE complex assembly contributes terminal. These findings suggest that α-Syn accumulation causes to clustering of SV at the AZ (Figure 5A). Besides functioning a toxic gain of function phenotype at the synapses, thus impairing as chaperone, α-Syn is also important for maintenance and their function and connectivity. redistribution of the SNARE complex which is directly implicated in neurotransmitter release, including dopamine (Burré et al., α 2010; Diao et al., 2013). Accumulated -Syn Impairs Rabs and Their The synaptic accumulation of oligomers and aggregated α- Function in Synaptic Vesicle Trafficking Syn and its interaction with components of the presynaptic The trafficking of synaptic vesicles within the presynaptic machinery, such as SNARE proteins, is likely to underlie at terminal is vital to recruit them from the SV pools into the active least one of the pathological mechanisms that lead to synaptic zone (Figure 4; Südhof, 2000). This is a complex process that dysfunction in the early stages of PD (Box 1) (Figure 5B). In involves a significant number of proteins and must be tightly support of this hypothesis, studies in vitro have demonstrated controlled (Takamori et al., 2006; Südhof, 2013b). Amongst the large α-Syn oligomers bind to the N-terminal domain of VAMP- proteins involved in presynaptic vesicle trafficking, a superfamily 2, thus blocking SNARE-mediated vesicle docking (Choi et al., of highly conserved small GTPases called Rab act as the master 2013), which in vivo could be directly linked to impaired regulator of intracellular trafficking processes, including vesicle exocytosis of neurotransmitters in overexpression models of α- budding and uncoating, motility, tethering and fusion (reviewed Syn. Interestingly, another in vitro study by Lai and co-authors in Kelly et al., 2012). Rab3A protein, for instance, is abundant in revealed that levels and different toxic species of α-Syn inhibit SV with its activity regulated by two effectors: Rabphilin and RIM

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FIGURE 5 | Onset of α-Syn-mediated synaptopathies is likely related to impaired presynaptic active zone function and defective neurotransmitter release. (A) In healthy subjects, the presynaptic terminal of a neuron comprises a functional exo-endocytotic cycling machinery, including vesicle pools, SNARE complex proteins and the active zone (AZ) which is formed of a dense network of proteins called AZ matrix. The AZ matrix is the site of action for synaptic vesicle tethering, docking and membrane fusion for neurotransmitter release which is mediated by SNARE complex proteins including VAMP-2, SNAP-25, and Syntaxin-1. This process is directed and co-chaperoned by CSP and α-Syn toward the AZ, a scaffold that includes ELKS/CAST proteins, the Drosophila homolog of Bruchpilot (BRP). BRP mutants have been shown to cause dramatic reduction of nerve-evoked transmission. Maintenance of BRP within the AZ matrix scaffold is dependent on direct interaction with NMNAT which protects BRP against ubiquitin (Ub)-mediated degradation and activity-induced neurodegeneration. (B) In PD patients, presynaptic neuronal terminals accumulate toxic oligomers and protofibrils of α-Syn that interfere with the exo-endocytotic cycling machinery, including AZ proteins NMNAT and BRP/ELKS/CAST (enlarged in C). (C) Disease-related downregulation of NMNAT leads to ubiquitination and degradation of BRP/ELKS/CAST, resulting in the gradual decrease and eventual dissolution of the AZ matrix, ultimately causing impaired synaptic transmission and α-Syn-mediated synaptopathy. However, it is currently unknown (doted red lines) whether toxic forms of α-Syn directly or indirectly interfere with NMNAT and/or BRP/ELKS/CAST, thereby causing the AZ to lose its physiological function.

(for Rab3 interacting molecules; Shirataki et al., 1993; Wang et al., were found to trap Rab3A and prevent its interaction with 1997). Mechanistically, Rab3A is part of a complex formed by the rabphilin, thus suggesting that exocytosis and neurotransmitter AZ proteins Munc13 and RIM, designed to capture SVs for final release are affected in Lewy body diseases (Dalfó et al., 2004). membrane attachment called tethering and to prepare them for Further studies have proposed that Rab3A regulates membrane synaptic release (Wang et al., 1997; Dulubova et al., 2005). association of α-Syn in a GTP-dependent manner (Chen et al., α-Syn was shown to interact and cause defects in vesicular 2013), implying functional integration of α-Syn into Rab proteins trafficking pathways including those taking place at the cycle between the cytosol and the membrane-bound state, and presynaptic terminal of neurons (Dalfó et al., 2004; Gitler et al., hence into the SV cycle (Bendor et al., 2013). 2008; Abeliovich and Gitler, 2016). In line with this observation, These functional interactions are further supported in a several members of Rab GTPase family have been identified Caenorhabditis elegans model overexpressing wild type human α- to interplay with α-Syn (Chutna et al., 2014; Gonçalves et al., Syn which revealed that manipulating levels of Rab3A, Rab1 and 2016). This interaction has also been observed in post-mortem Rab8A significantly rescued α-Syn mediated loss of DA neurons tissue of DLB and MSA patients with LB pathology where α-Syn (Gitler et al., 2008). Similar effects were seen in rat midbrain abnormally interacts with Rab3A. In addition, α-Syn aggregates cultures transfected with A53T mutant α-Syn (Gitler et al., 2008).

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α-Syn mediated neurotoxicity was hypothesized to be caused by effects caused by toxic α-Syn, such as alterations of SV α-Syn affecting transport of SV via direct interaction with the pools, SNAREs, and intracellular trafficking that together lead trafficking machinery (Gitler et al., 2008). Furthermore, studies to impaired neurotransmitter exocytosis (Larsen et al., 2006; in Drosophila melanogaster suggest that Rab3 is also involved in Nemani et al., 2010; Scott et al., 2010; Janezic et al., 2013). the formation of the AZ architecture, however the mechanisms Furthermore, the accumulation of toxic α-Syn species in the AZ remain unknown (Chen et al., 2015; Bae et al., 2016). triggers the proteasome system to degrade not only α-Syn but also AZ matrix components, the levels of which are normally The Active Zone Matrix and α-Syn regulated by proteasome activity (Wang et al., 2017). The docking and fusion of SVs are restricted to the active Work carried out in Drosophila melanogaster has provided zone, demonstrating that the AZ is essential to translate at least one mechanistic pathway by which the presynaptic an incoming action potential into neurotransmitter release AZ is sustained under disease-related stress (Figure 5). The (Figure 4; Schoch and Gundelfinger, 2006; Südhof, 2012; Wang maintenance of the AZ structure against activity-induced et al., 2016). The AZ is a highly conserved specialized degeneration is provided by a chaperone called Drosophila presynaptic scaffold characterized by a dense network of nicotinamide mononucleotide adenylyltransferase (dNMNAT) proteins called AZ matrix. In mammals, this matrix includes (Zang et al., 2013). This neuroprotective effect is provided RIM, ELKS, Munc13, Bassoon/Piccolo, Rim-BP, and Liprin-α through NMNAT, which protects the AZ protein Bruchpilot (Schoch and Gundelfinger, 2006; Südhof, 2012; Wang et al., (BRP), a homolog of mammalian ELKS/CAST proteins, from 2016). Furthermore, additional proteins are present but not ubiquitination and subsequent degradation. However, it is not restricted to the AZ, including SNAREs, ion channels, receptors, known whether high levels and/or toxic α-Syn species, such as cytoskeletal proteins and adhesion molecules (Wang et al., oligomers and fibrils, exert a toxic gain of function onto the AZ 2016). Considering the high number of proteins constituting by interfering with NMNAT function (Figures 5B,C). the AZ, it is important for this presynaptic compartment to Unlike Drosophila that only encodes one NMNAT homolog, be tightly regulated according to synaptic activity for reliable mammals possess three different NMNAT orthologues that neurotransmitter release to occur (Körber and Kuner, 2016). are distinguishable by their differential localization within Although a large amount of evidence indicates that subcellular compartments (Brazill et al., 2017). NMNAT1 is a accumulation of α-Syn at the presynaptic terminal causes nuclear protein, NMNAT2 is localized to the Golgi apparatus reduction of proteins involved in synaptic transmission, to our and NMNAT 3 is predominantly mitochondrial (Ali et al., 2013). knowledge, α-Syn has not been shown to directly interact with NMNATs are classically known for their enzymatic function AZ matrix proteins under normal conditions. In addition to that, of catalyzing NAD+ synthesis, which is an essential cofactor only a few studies have explored the pathological role of α-Syn in many cellular processes including oxidative reactions and affecting proteins associated with the AZ. Mantin and colleagues transcriptional regulation (Lau et al., 2009). NMNAT is also able reported mice overexpressing human mutant A53T α-Syn which to act as a molecular chaperone (reviewed in Ali et al., 2013), showed abnormal inclusions of α-Syn within dendrites and in which is critical for neuronal homeostasis and protection against the presynaptic AZ (Martin et al., 2006). Furthermore, a recent cellular stress (Ali et al., 2012; Ocampo et al., 2013; Rallis et al., ultrastructural study by Kohl and colleagues utilized a bacterial 2013). However, this role does not require the enzymatic function artificial (BAC) rat model expressing full-length of NMNAT, as the inactive form of dNMNAT was able to protect human SNCA gene. These transgenic rats displayed extended Drosophila photoreceptors against activity-induced degeneration AZs in synapses of hippocampal CA3 neurons compared to (Zhai et al., 2006, 2008). non-transgenic animals, a phenotype that was accompanied by Consistent with the role of NMNAT as a neuronal reduction of the postsynaptic density. However, PSD95 levels maintenance factor, it has been shown that NNMAT2, which were found to be upregulated in the dentate gyrus/CA3 regions is enriched in synaptic terminals and SVs, is downregulated in combination with downregulation of synapsin1 and RIM3 in several degenerative disorders. These include PD, AD, (Kohl et al., 2016). This study suggests that α-Syn does not only Huntington’s disease (HD) and frontotemporal lobar compromise the presynaptic but also postsynaptic terminals by degeneration with ubiquitin- and TDP-43-positive inclusions altering both RIM3 and PSD95. The RIM protein family is an (FTLD-TDP) (Ali et al., 2013). NMNAT2 alterations were also essential regulator of active zone function (Mittelstaedt et al., found in a mouse model of tauopathy expressing mis-sense 2010; Südhof and Rizo, 2011). Another AZ protein called Piccolo mutation P301L, found in some frontotemporal dementia was shown to be absent in a significant number of synaptic and parkinsonism linked to chromosome 17 (FTDP-17) boutons in a model overexpressing α-Syn (Scott et al., 2010). cases (Ljungberg et al., 2012). The neuroprotective effects of Piccolo along with Bassoon are specific to the vertebrate AZ and mammalian NMNAT2 has been explored in more detail in do appear to have a major function in guiding SVs to the AZ axonal degeneration, where loss of NMNAT2 is considered to be (Mukherjee et al., 2010; Südhof, 2012). a physiological stimulus in severed axons and their subsequent Since α-Syn has been demonstrated to accumulate at the AZ Wallerian degeneration (Gilley and Coleman, 2010). In line with (Martin et al., 2006), it is reasonable to hypothesize that AZ this notion, NMNAT2 overexpression was found to delay injury- matrix proteins become dysfunctional over time. Moreover, α- induced axonal degeneration in zebrafish (Feng et al., 2010) and Syn accumulation in the AZ may jeopardize a compensatory to protect mice against tauopathy-induced neurodegeneration activity of AZ proteins to overcome the upstream deleterious (Ljungberg et al., 2012).

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Accumulated α-Syn Affects Synaptic the misregulation of several synaptic proteins (Table 1), leading Vesicle Endocytosis to functional impairment of the presynaptic terminal in both In order to sustain high rates of synaptic transmission without animal models and PD patients (Figure 5). These alterations depleting the supply of SVs, neurons have to rely on efficient at the presynapse have been shown to be progressive and endocytic recycling of SV membranes (Figure 4; Saheki and selective to dopaminergic neurons, at least in mouse models De Camilli, 2012). The synuclein family is required for SV (Volpicelli-Daley et al., 2011; Janezic et al., 2013). Thus, upon endocytosis (Vargas et al., 2014). Using a triple synuclein α-Syn accumulation, DA neurons show reduced firing rates, a KO mouse model, Vargas and colleagues demonstrated that phenotype that positively correlates with behavior impairment, are required for fast and efficient kinetics of early ultimately followed by degeneration of these neurons (Janezic steps of SV endocytosis, membrane bending and cargo selection et al., 2013). (Vargas et al., 2014). Fundamentally, the impaired endocytic A similar sequence of events whereby presynaptic deficits process observed in the triple KO mouse could be re-established precede neuronal loss are also recapitulated in induced by individual expression of α-, β-, or È-Syn, demonstrating they pluripotent stem cells (iPSCs) derived from PD patients that are functionally redundant in the endocytic process (Vargas et al., harbor the A53T α-Syn mutation. Kouroupi and colleagues 2014). observed a significant downregulation of genes coding for Rab5, which has been localized to the early endosome and presynptic proteins in comparison to controls (Kouroupi et al., is implicated in the regulation of neuronal endocytosis in 2017). Strikingly, A53T α-Syn iPSCs cells exhibited thinner, association with Rabaptin-5 (Stenmark et al., 1994; Cataldo less fasciculated neuronal processes that were accompanied by et al., 2000), appears to play a role in the internalization of a 27% reduction in the number of synaptic contacts. The exogenous α-Syn. Overexpression of constitutively active Rab5 or deleterious effects caused by overexpression of human α-Syn its effector Rabaptin-5 in cultured neurons lead to the formation were also observed in Drosophila which identified reduced of intracellular inclusions resembling Lewy bodies (Sung et al., synaptic connectivity between DA neurons and Kenyon cells 2001). More recently, a study revealed that high levels of α-Syn even before the onset of motor abnormalities (Riemensperger promotes its interaction with dynein, a retrograde motor protein, et al., 2013). inducing a significant increase in activated levels of Rab5 and Together this data suggests that there is a potential Rab7, both key regulators of the endocytic process (Fang et al., direct association, between α-Syn accumulation and dopamine 2017). metabolism, which in turn makes DA neurons especially Under intense electric stimulation, overexpression of wild vulnerable to cellular stress and subsequent neurodegeneration. type α-Syn in lamprey nerve terminals caused loss of SVs Indeed, several recent findings support this hypothesis, which and an expanded plasma membrane which pointed toward could shed light onto the pathogenic mechanisms underlying DA impaired vesicle recycling (Busch et al., 2014). However, this neuron-specific cell death in PD. endocytic deficit was not observed when low stimulation was applied. Interestingly, overexpression of mutant A30P α-Syn, α with a disrupted N-terminal that greatly impairs its binding to -SYN AND DOPAMINE: A DANGEROUS phospholipids and disrupt the α-helical conformation, showed LIAISON very little effect on vesicle recycling. Therefore, Busch and colleagues concluded that synaptic endocytic failure could only The loss of dopaminergic neurons in the substantia nigra be visualized when high synaptic activity is in place and along with α-Syn enriched Lewy body inclusions are the main under proper folding of α-Syn N-terminal α-helix (Busch histopathological hallmarks of PD (Lees et al., 2009). DA et al., 2014). Corroborating these findings, overexpression of neurons are thought to be the most vulnerable cell type in mutant human A53T α-Syn, but not A30P α-Syn, at the PD, which led to several hypotheses attempting to explain calyx of Held terminals in mice was also shown to cause the underlying mechanisms. One of these is related to the impairment of slow and rapid endocytosis, and restoration of high-energy requirement of DA neurons due to their poorly the RRP (Xu et al., 2016). These studies endorse the role of α- myelinated axonal arborisation which is orders of magnitude Syn in membrane curvature formation and stabilization, and larger than any other neuronal cell type (Pissadaki and Bolam, consequently for endocytosis (Pranke et al., 2011; Busch et al., 2013). This hypothesis predicts that any disturbance inducing 2014). a negative energy balance would be especially dangerous to DA neurons, placing them at high risk in case of energy failure, such α as mitochondrial dysfunction, which has been shown to play an -Syn Related Presynaptic Deficits important role in PD (Lin and Beal, 2006). Converge in Dopaminergic Neurons Although energy balance contributes to the vulnerability of Under physiological conditions, native soluble α-Syn is involved DA neurons, regulation of dopamine levels at the synaptic in several steps in the presynaptic terminal that are required terminals is thought to be vital for DA neuron susceptibility to trigger exocytosis upon activation by an action potential observed in PD (Lotharius and Brundin, 2002b). Accordingly, (Burré, 2015). However, these steps are not exclusively regulated α-Syn has been implicated as a key regulator of dopamine by α-Syn. Under pathological conditions, the presence of toxic homeostasis (Abeliovich et al., 2000; Venda et al., 2010). α-Syn species such as oligomers and amyloid fibrils, triggers The activity of the rate-limiting enzyme tyrosine hydroxylase

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(TH) responsible for producing dopamine through conversion VMAT2 were also observed in synaptic vesicles isolated from of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), is post-mortem brain tissue of PD patients, including reduction negatively regulated by increased concentration of α-Syn in vitro in vesicular uptake and the binding ability of VMAT2 (Pifl (Perez et al., 2002). As a result, reduced levels of dopamine were et al., 2014). These findings are in agreement with previous observed in cell culture transfected with A53T mutant α-Syn studies showing that increased levels of α-Syn inhibit VMAT2 (Perez et al., 2002). activity, ultimately increasing cytosolic levels of dopamine (Guo In line with these findings, reduced TH activity was found in et al., 2008). As a consequence, dysregulation of dopamine mouse models overexpressing α-Syn (Masliah et al., 2000; Kirik transport caused by impaired DAT and VMAT2 activity will et al., 2002). Mechanistically, α-Syn decreases TH activity by lead to altered levels of dopamine (Lotharius and Brundin, reducing its phosphorylation in vitro (Perez et al., 2002; Peng 2002b; Nutt et al., 2004). Dopamine by itself is a risk factor et al., 2005) as TH is activated by phosphorylation of seryl for dopaminergic neurodegeneration; it is highly sensitive to residues in the TH regulatory domain (Alerte et al., 2008). In spontaneous degradation and to produce quinone as well as turn, by silencing α-Syn in MN9D cells, increased TH activity other reactive oxygen species until it is sequestered into vesicles and dopamine biosynthesis were observed (Liu et al., 2008). In (Lotharius and Brundin, 2002a,b; Xu et al., 2002; Caudle et al., addition to the ability of α-Syn to regulate TH activity, work 2007). performed by Tehranian and colleagues demonstrated that α-Syn This data suggests an intricate and potentially direct interplay can interact with L-aromatic amino acid decarboxylase (AADC) between α-Syn and dopamine that directly contributes to and can reduce its activity (Tehranian et al., 2006). AADC acts functional deficits of DA neurons. In support of this, Mor in the last step of dopamine synthesis by converting L-DOPA to et al. recently demonstrated that dopamine promotes α- dopamine (Tehranian et al., 2006). Syn oligomerisation in vivo and that disrupting the ability α-Syn does not only modulate the activity of enzymes of dopamine to stabilize or modify α-Syn oligomers was responsible for dopamine synthesis, but it also impairs transport sufficient to rescue dopamine-mediated toxicity (Mor et al., and uptake of dopamine by altering the activity of the 2017). These results recapitulate in vitro data revealing the vesicular monoamine transporter 2 (VMAT2) and the dopamine ability of dopamine to trigger formation of α-Syn oligomers transporter (DAT), respectively. VMAT2 is responsible for (Choi et al., 2013). This cooperative toxicity of α-Syn and dopamine uptake from the cytoplasm into SV (Lotharius and dopamine was also recapitulated in C. elegans (Mor et al., Brundin, 2002b). DAT transports dopamine from the synaptic 2017). Furthermore, a corresponding mouse model revealed cleft back into the cytoplasm is the primary mechanism and early mild synaptic defects in the striatum without degeneration possibly the most important regulator of extracellular dopamine of cell bodies; however, by 5 months post-injection, the mice concentrations (Vaughan and Foster, 2013). Lundblad and exhibit a severe loss of dopaminergic nerve terminals (−62% colleagues showed that unilateral overexpression of human wild VMAT2 and −55% DAT) as compared to only 25% loss of type α-Syn in the SN of adult rats caused 50% reduction dopaminergic neurons (Mor et al., 2017). These results reinforce in dopamine reuptake without axonal damage 10 days after the hypothesis that α-Syn mediated early dopamine imbalance exposure (Lundblad et al., 2012). Over time, dopamine reuptake at synaptic terminals is the initiating event which in turn was further decreased in the striatum, followed by axonal swelling triggers synaptic and axonal dysfunction, subsequently leading to and dystrophic axons in the presence of α-Syn aggregates degeneration of nigrostriatal pathways and DA neuron-specific and a profound 70% reduction in dopamine release upon cell death. chemical pulse with potassium chloride (Lundblad et al., 2012). Additionally, α-Syn was shown to interact with DAT and in turn to form complexes via the carboxyl-terminal tail of DAT and CONCLUSIONS the NAC domain of α-Syn (Lee et al., 2001). This interaction and complex formation reduced DAT activity due to impairment Studies in PD-related animal models and post-mortem patient of dopamine reuptake from the synaptic cleft (Wersinger and material demonstrate that soluble oligomers and aggregating Sidhu, 2003; Wersinger et al., 2003), suggesting that α-Syn protofibrils of α-Syn accumulate in synapses and axons prior accumulation causes a vicious cycle of impaired VMAT2 and to the onset of disease symptoms. These accumulating toxic DAT function. α-Syn species impair synaptic compartments and presynaptic Consistent with this notion, Bellucci et al. showed that processes required for neuronal communication, including mice expressing the truncated α-syn120 exhibited marked maintenance of SV pools, SV trafficking, tethering, docking, redistribution of DAT/α-Syn complexes in the striatum and priming and fusion of SV within the plasma membrane at the the SN (Bellucci et al., 2011). These findings suggest that active zone. The resulting synaptopathy phenotypes manifest α-Syn acts as a synaptic modulator of DA in early PD prior to dopaminergic neurodegeneration, hence reinforcing pathogenesis by regulating the subcellular distribution of key the hypothesis that onset and evolution of PD progress in proteins such as DAT (Bellucci et al., 2011). DAT has also a dying back-like manner, from initial dysfunction at the been reported to be absent in the putamen of PD patients synapse and impairing axonal connections, to eventual neuronal (Seeman and Niznik, 1990). Interestingly, VMAT2 was shown cell body death. Although α-Syn is expressed throughout the to be incorporated into Lewy bodies of SN neurons in PD brain and enriched at presynaptic terminals, DA neurons are patients (Yamamoto et al., 2006). Moreover, profound defects in most susceptible to toxic effects exerted by accumulating and

Frontiers in Neuroscience | www.frontiersin.org 13 February 2018 | Volume 12 | Article 80 Bridi and Hirth Mechanisms of α-Synuclein Induced Synaptopathy dysfunctional α-Syn. This fatal attraction is likely related to AUTHOR CONTRIBUTIONS α-Syn regulating dopamine levels in two distinct manners. First, α-Syn is a negative modulator of dopamine synthesis All authors listed have made a substantial, direct and intellectual by interplay with the rate-limiting enzyme TH and AADC. contribution to the work, and approved it for publication. Second, α-Syn disrupts VMAT2 and DAT activity and their function in regulating dopamine levels in the synaptic terminal. ACKNOWLEDGMENTS In turn, dysregulated dopamine levels fuel the formation of soluble α-Syn oligomers that are considered to be the We thank D. P. Srivastava, R. B. Parsons, D. Aarsland, A. most toxic species. This fatal interplay and its ensuing Perry, and W. H. Au for comments on the manuscript and B. vicious cycle of dopamine and α-Syn interference is likely Kottler for helpful suggestions. This work was supported by a the starting point driving synaptic dysfunction and impaired PhD fellowship from CAPES Foundation–Ministry of Education neuronal communication observed in the early, prodromal stages of Brazil to JCB and by the UK Medical Research Council of PD. (G0701498; MR/L010666/1) to FH.

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